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Group I & II intron

Group I & II intron

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ROLANDSALDANHA,
GEORGMOHR
MARLENE
BELFORT’
AND
ALANM.LAMBOWITZ,i
DepartmentsofMolecularGenetics
and
Biochemistry,and
theBiotechnologyCenter,TheOhioState
Universit
Columbus,Ohio43210,
USA;and
tMolarGeneticsProgram,WadsworthCenterforLaboratoriesandResearch,
New
York
State
DepartmentofHealth,Albany,New
York
12201-0509,
USA
GroupIandgroupII
introns
0892-6638/93/0007-0015/$01.50.©FASEB15
ABSTRACTGroupIandgroup
IIintrons
are
two
typesofRNAenzymes,ribozymes,thatcatalyzetheir
ownsplicingbydifferentmechanisms.Inthisreview,we
summarizecurrentinformationaboutthestructures
of
groupI
and
group
II
introns,
theirRNA-catalyzedreac-tions,thefacilitationofRNA-catalyzedsplicingbypro-teinfactors,andtheabilityoftheintronstofunctionasmobileelements.TheRNA-basedenzymaticreactionsand
intronmobilityprovideaframeworkforconsidering
theroleofprimordialcatalyticRNAsinevolutionandtheoriginofintronsinhigherorganisms.-Saldanha,R.,Mohr,G.,Belfort,M.,andLambowitz,A.M.GroupIand
group
IIintrons.
FASEBJ.7:
15-24;1993.
KyWords:catalyticRNAmobileint,vnsite-specificendonucleosereversetranscriptaseevolution
GROUPIINTRONSGroupIintronsarepresentinrRNA,tRNA,andprotein-codinggenes.TheyareparticularlyabundantinfungalandplantmitochondrialDNAs(mtDNAs),2buthavealsobeenfoundinnuclearrRNAgenesof
Tetrahymena
andotherlowereukaryotes,inchloroplastDNAs(ctDNAs),inbacteri-ophage,andrecentlyinseveraltRNAgenesineubacteria(seerefs1-3).MostgroupIintronshaveahighlyvariabledistribution,eveninrelatedorganisms,apparentlyreflectingtheirdispersalasmobileelements.Ontheotherhand,thesamegroupIintronispresentinthetRNAgenesofctDNAsandfivedifferentcyanobacterialspecies,suggestingthatitexistedbeforetheevolutionofplastidsandcouldbe1to3.5billionyearsold.ThefindingthatsomegroupIin-tronsareancientisconsistentwiththeideathattheyareremnantsofaprimordialRNAworld.Indeed,thecatalyticactivitiesofgroupIintronshavebeguntoprovideinsightintohowprimitiveRNAscouldhavecatalyzedtheirownreplication
and
contributedtotheevolutionofproteinsyn-thesis.SplicingmechanismandstructureAsfirstshownforthe
Tetrahymena
largerRNAintron,groupIintronssplicebyamechanisminvolvingtwotransesterifica-tionreactionsinitiatedbynucleophilicattackofguanosineatthe5splicesite(Fig.1)(4).Theremarkablefindingforthe
Tetrahymena
intronwasthatsplicingrequiresonlyguanosineandMg.Becausebondformationandcleavagearecou-pled,splicingrequiresnoexternalenergysourceandiscom-pletelyreversible.Afterexcision,somegroupIintronscir-cularizeviaanadditionaltransesterification,whichmaycontributetoshiftingtheequilibriuminfavorofsplicedproducts(4).TheabilityofgroupIintronstocatalyzetheirownsplic-ingisrelatedtotheirhighlyconservedsecondaryandter-tiarystructures(Fig.2)(1,5,6).Asinproteinenzymes,thefoldingoftheintronresultsintheformationofanactivesitejuxtaposingkeyresiduesthatarewidelyseparatedinprimarysequence.ThisRNAstructurecatalyzessplicingbybringingthe5and3splicesitesandguanosineintoprox-imityandbyactivatingthephosphodiesterbondsatthesplicesites(4).DifferentgroupIintronshaverelativelylittlesequencesimilarity,butallshareaseriesoftheshort,con-servedsequenceelementsP,
Q,
R,
andS,
withpartsofP/QandR/Sbasepairingintheconservedstructure(Fig.2A).TheboundariesofgroupIintronsaremarkedsimplybyaUresidueatthe3endofthe5exonandaGresidueatthe3endoftheintron(5,6).TheconservedgroupIintronsecondarystructurewasdeducedfromphylogeneticcomparisons,andspecificfea-tureshavebeenconfirmedbyanalysisofinvivoandinvitromutationsandbystructuremapping(1,5,6).Thestructure,showninFig.2A,consistsofaseriesofpairedregions,denotedP1-PlO,separatedbysingle-strandedregions(denoted
J)
orcappedbyloops(denotedL).P1andPlO,whichcontainthe5
and
3splicesites,respectively,areformedbybasepairingbetweenaninternalguidesequence(IGS),generallylocatedjustdownstreamofthe5splicesite,andexonsequencesflankingthesplicesites.GroupIintronshavebeenclassifiedintofourmajorsubgroups,designatedIAtoID(I),basedondistinctivestructuralandsequencefeatures.GroupIAintrons,forexample,containtwoextrapairings,P7.l/P7.laorP7.l/P7.2,betweenP3andP7,whereasmanygrouplBandICintronshavealargeexten-sionofP5,termedP5abc.Individualintronsmaycontainadditionalsequences,includingopenreadingframes(ORF5),inpositionsthatdonotdisrupttheconservedcorestructure.Theregionofthe
Tetrahymena
intronrequiredforen-zymaticactivity,thecatalyticcore,consistsofP3,P4,P6,P7,P8,andP9.0(1).StudiesusingFe(II)-EDTA,areagentthatcleavesthesugar-phosphatebackbone,haveshownthatpartsofthecoreareburiedinthestructureinaccessibletothesolvent,thatMg2isnecessaryforfoldingoftheintron,andthatindividualRNAdomainsfoldinaspecificorderasMg2isincreased(4,7).
All
groupIintronshavefundamen-tallysimilarcorestructures,butsubgroup-specificstructuressuchasP7.1,P7.2,andP5abcappeartoparticipateinaddi-Towhomcorrespondenceshouldbeaddressed,at:DepartmentofMolecularGenetics,TheOhioStateUniversity,484WestTwelfthAve.,Columbus,OH43210,USA.2Abbreviations:ctDNA,chioroplastDNA;EBS,exonbindingsite;IBS,intronbindingsite;IGS,internalguidesequence;LTR,longterminalrepeat;mtDNA,mitochondrialDNA;ORF,openreadingframe;aaRS,aminoacyl-tRNAsynthetase;RT,reversetranscriptase.Toaccommodatealimitationofthenumberofreferences,wehavecitedreviewswhereverpossible.
 
5Exon
Intron
3Exon5Exon
muon
3Exon
UGGUGCGAAY3
3.j
-
I
Intron
3Exon
CExon
5Exon
I,
+
5
Exon
,2wcccc?cclU3.OH
I,
5
Exon3Exon
U
+
G
GroupIIntron
16Vol.7January1993TheFASEBJournalSALDANHAETAL.
5Exon3Exon
wmnn
+
CL
GroupHIntron
Figure
LSplicingmechanismsofgroupIandgroupIIintrons.Conservednucleotidesinintronsandflankingexonsareindicated.tionalinteractionsthatstabilizethecorestructureindiffer-entways(1,8).Athree-dimensionalmodelofthegroupIintroncatalyticcorehasbeendevelopedbyMichelandWesthof(1)(Fig.2B,C).Theunderlyingassumption,firstsuggestedbyKimandCech(9),isthatadjoininghelicalsegmentsstackcoaxiallytocreatetwoextendedhelices,P6a-P6-P4-P5andP8-P3-P7,whichformacleftcontainingtheintronsactivesite(Fig.2B,C).IntheMichel-Westhofmodel,therelativeorientationofthetwohelicesisconstrainedbyapreviouslyproposedtriplehelixinvolvingpartsofJ3/4-P4-P6-J6/7andbypotentialter-tiaryinteractionsidentifiedbycovariationofnucleotidesthatarenotaccountedforbysecondarystructure.Anumberofthesepredictedinteractionsinvolvepurine-richloopsorbulgesengagedinlong-rangeinteractionswithdoublehelices(Fig.2B).Theactivesiteoftheintron,formedbythecleftbetweenthetwohelices,containsbindingsitesfortheguanosinecofactorandP1andPlOcontainingthe5and3splicesites.Themodelisdesignedsothatthedispositionofthesebindingsitesaccountsfortheknownsplicingmechan-ism,whichrequiresappropriatealignmentsofguanosineandthe5
and
3exonsinthefirstandsecondstepsofsplic-ing(4).DeoxynucleotideandphosphorothioatesubstitutionexperimentssuggestthatfunctionallyimportantMg2ionsarecoordinatedatspecificpositionsaroundtheactivesite(e.g.,P1andJ8/7),wheretheymayfunctiondirectlyinphos-phodiesterbondcleavage(1,10).Basicfeaturesofthepredictedthree-dimensionalstructurehavebeensupportedbymutantanalysisinvitroandbytheuseofspecificallyposi-tionedphotochemicalcross-linkingandaffinitycleavagerea-gents(1,11,12).DefinitionofsplicesitesandbindingtotheintroncoreThe5and3splicesitesofgroupIintronsaresubstratesthatareactedonbythecatalyticcore,andtheycanberecog-nizedandcleavedbythecorewhenaddedonseparateRNAmolecules(4).The5splicesiteisdefinedbytheP1pairingbetweenthelOSandthe5exon.NeitherthesequencenorlengthofP1isfixed,buttheconservedUatthe3endofthe5exonalwaysformsawobblebasepairwitha0residueintheIGS(Fig.2A).AnalysisofinvitromutantsshowedthatthedistanceoftheUGpairfromthebottomoftheP1helixiscriticalforefficientcleavageinthe
Tetrahymena
intron(4,13)andthatJl/2andP2alsoplayaroleinthepositioningofP1relativetothecore(1,14,15).IntheMichel-Westhofmodel,P1ispredictedtobindtothecoreviatertiaryinteractionswithJ8/7andJ4/5(Fig.
2B)
(1).Experimentshaveshownthatthe2OHsatP1positions-2and-3contributeenergeticallytobindingP1tothecore,andthatthe2OHat-3interactswithaspecificresidueinJ8/7.ThelatterinteractionissomewhatdifferentfromthatproposedbyMichelandWesthof(1),butwasreadilyaccom-modatedbyalocalrevisionofthemodel(12).Thepositioningofthe3splicesiteingroupIintronsde-pendsonatleastthreeinteractions,whoserelativeimpor-tancediffersindifferentintrons(4).Thesearethe
PlO
pair-ingbetweenthelOSandthe3exon,bindingoftheconserved0residueatthe3endoftheintrontothe0-bindingsiteinthesecondstepofsplicing,andanadditionalinteraction,P9.0,whichinvolvesbasepairingbetweenthetwonucleotidesprecedingtheterminal0oftheintronandtwonucleotidesinJ7/9(Fig.2A).Guanosine-bindingsiteGroupIintronshave
Km
valuesforguanosinethatareaslowas1
eM
andreadilydiscriminatebetweenguanosineandothernucleosides(4).Themajorcomponentoftheguanosine-bindingsitecorrespondstoauniversallycon-servedGCpairinP7(Fig.2A)(1,4).Guanosinewasinitiallyproposedtointeractwiththisbasepairviaformationofabasetriple,butthecontributionofneighboringnucleotidesandthebindingofanalogsarealsoconsistentwithamodelinwhichguanosinebindsaxiallytotheconserved0andflankingnucleotides(16).Theguanosine-bindingsiteofgroupIintronscanalsobeoccupiedbytheguanidinogroupsofarginineorantibiotics,suchasstreptomycin,whichactascompetitiveinhibitorsofsplicing(17).Yarus(18)notedthatthethreenucleotidesthatconstitutetheguanosine-bindingsiteindifferentintrons,AGA/GandCGA/G,correspondtoArgcodons,andspeculatedthatrecognitionofaminoacidsbythisandotherfunctionallyim-portantsitesincatalyticRNAscouldhaveplayedaroleintheevolutionofthegeneticcode.
OtherreactionscatalyzedbygroupIintrons
Inadditiontosplicing,groupIribozymescancatalyzeavar-ietyofintermolecularreactions,includingendonucleolyticcleavageofRNAandDNA,RNApolymerization,nucleo-tidetransfer,templatedRNAligation,andaminoacyl-estercleavage(4,19).Allthesereactionsusethesameactivesiteasthesplicingreaction,andtheycanbegeneralizedasshowninFig.3A.Intheforwarddirection,equivalenttothefirststepofsplicing,thereactionisthinucleophilicattackofguanosineonthephosphodiesterbndsucceeding
XXU
(whereXXdenoteexonnucleotidesthatpairwiththeIGS[XX]toformP1).Inthereversediretion,equivalenttothesecondstepofsplicing,thereactionisnucleophilicattackofthe3OHofXXUonthephosphodiesterbond3oftheter-minal0oftheintron.Intheintermolecularreactions,thesubstratesareoligonucleotidesthatareeithercognatesofP1
 
LI
L2
P1
I’ll’’’
L8
P8
S
BCA
L5
GROUPIANDGROUPIIINTRONS
17
Figure2.Group
I
intronstructure.
A)
Conservedsecondarystructure.Arrowsindicatesplicesites.ConservedsequencesP,
Q,
R,andS(5,6)areindicatedbyheavylines.Asteriskdenotesguanosine-bindingsite.Dashesindicatebasepairs.Dotindicateswobblebasepairatthe5splicesite.NucleotidesinvolvedinP9.0pairingareindicatedasNNandNN.NucleotidesinvolvedinPlOpairingareindicatedasMMMandMMM.IGS,internalguidesequence.
B)
Schematicillustratingsomeproposedtertiaryinteractionsintheyeastwintron(1,8).Interactingnucleotidesareconnectedbydashedlines.CircledGshowsfreeguanosineinteractingwithconservedGCpairoftheguanosine-bindingsite.
C)
Three-dimensionalstructuralmodelofthe
Tetrahymena
largerRNAintronfromMichelandWesthof(1),reproducedbypermissionofthe
JournalofMolecularBiology.
orbasepairwiththelOStoreconstituteanalogsofP1orPlO.Inthesite-specificendonucleasereaction,anRNAsub-stratethatresemblesthe5exonandbasepairstotheIGSiscleavedbyguanosine(Fig.
3B)
orOH-(notshown)inaprocessanalogoustothefirststepofsplicing.Thisreactionhasbeenmodifiedinvariouswaysthatillustratetheversatil-ityoftheribozyme.Therate-limitingstepisthereleaseofthecleavedRNAproducts,sothatmutationsinthelOSthatweakenRNAbindingincreasetherateofreaction(20).Thesubstratespecificityofthereactionisdeterminedbythese-quenceoftheIGS,whichcanbechangedtoenabletheribo-zymetocleaveotherRNAsubstrates.DNAiscleavedinefficiently,butribozymeshavingenhancedDNAcleavageactivityhavebeenobtainedbyiterativeselection(21,22).Recently,the
Telrahymena
ribozymewasshowntocatalyzethereverseofanaminoacylationreaction,hydrolysisofanaminoacyl-ester,N-formyl-L-methionine,attachedtotheCCAendofanoligonucleotidethatbasepairstoanap-propriatelymodifiedIGSsequence(Fig.
3E)
(19).Althoughinefficient,thisreactiondemonstratesthattheribozymecanactoncarboncenters,
and
supportsthehypothesisthatan-cientcatalyticRNAscouldhavefunctionedasaminoacyl-tRNAsynthetasesintheearlyevolutionofproteinsynthesis.InsupportoftheideathatprimitiveRNAscouldhavebeenthefirstself-replicatingmolecules,groupIribozymeshavebeenshowntoactasRNApolymerases.Initially,non-templatedadditionofnucleotidestooligonucleotidesbound

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